This invention relates to a method and means for separating radiation of one wavelength from radiation of a different wavelength. In various situations there is need to separate and measure radiation of one wavelength which is mixed with radiation of a different wavelength. One such situation is presented when it is desired to measure infrared radiation which is mixed with radiation in the visible range. A situation in which the invention is particularly useful is presented when it is desired to measure fluorescent radiation which is mixed with residual excitation radiation of a different wavelength.
In the use of conventional fluorescent radiation measurement instrumentation, a fluorescent analyte sample in a cuvette is placed in a beam of monochromatic light, and the resultant absorption of radiant energy by the molecules of the analyte sample raises the vibration level of such molecules from the ground state to one of the excited electronic levels. The absorption step occurs within 10.sup.-15 seconds, and fluorescence results from the spontaneous radiative transition that occurs when the molecules of the analyte sample return to the ground electronic state upon termination of exposure to the incident radiation. The resulting fluorescent light is given off equally in all directions at a wavelength different from that of the exciting light. In terms of intensity measured in photons, the incident light is generally orders of magnitude greater than the emitted fluorescent light, for example of the order of 10,000 to 1 or greater.
In most of the instrumentation available for measurement of fluorescent radiation, the emitted fluorescent radiation is viewed from a direction perpendicular to the incident excitation beam. This geometry minimizes the effect of light scattering by the solution and cell; however, only a very small percentage of the fluorescent light reaches the detector. Since the detector is unable to distinguish between the incident and the fluorescent light, interference filters are used which are intended to prevent residual radiation of the wavelength of the incident beams from reaching the detector, while permitting radiation of the wavelength of the emitted fluorescent radiation to reach the detector.
The need to more effectively collect fluorescent radiation has lead to the development of the so-called integrating sphere-type fluorimeter, such as that shown by W. R. Ware and W. Rothman, in Chem. Phys. Letters 39 (1976) 449. In that instrument the cuvette is located centrally of the integrating sphere and the fluorescent radiation emitted by the sample is reflected by the sphere walls until it is absorbed by a photodetector or lost through the incident beam entrance opening.
While the use of the integrating sphere results in the collection and detection of a substantially greater quantum of fluorescent radiation than was possible with the prior instruments referred to herein, the accuracy of the measurement of such fluorescent radiation is impaired by the fact that, despite the presence of interference filters, interference between residual incident light and the fluorescent light nevertheless occurs. This situation exists because presently available filters are simply unable effectively to screen out all of the residual incident light.